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Xanthones from the tropical fruit mangosteen (Garcinia mangostana) display anti-inflammatory and anti-oxidative activities. Here, we isolate and identify potential inducers of the aryl hydrocarbon receptor (AhR) and nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathways from mangosteen using a bioassay-guided strategy. Mangosteen fruit pericarp extracts were subjected to sequential solvent extractions, followed by chromatography coupled with NMR spectroscopy and mass spectrometric analyses for identification and isolation of pure compounds. Isolation of active fractions led to seven prenylated xanthones that were identified and subsequently evaluated for bioactivity. In vitro luciferase reporter cellular assays using H1L6.1c3 (AhR induction) and HepG2-ARE (Nrf2 induction) were used to identify AhR and Nrf2 activators. All seven prenylated xanthones displayed AhR inducing activity, whereas only five xanthones activated Nrf2. Garcinone D (GarD) significantly upregulated AhR/Cyp1a1 and Nrf2/HO-1 protein expression and enhanced zonula occludens-1 and occludin protein levels in HT-29 cells. In addition, GarD inhibited oxidative stress-induced intestinal epithelial barrier dysfunction by enhancing tight junction (TJ) proteins and inhibition of reactive oxygen species production. Inhibition of AhR by pretreating cells with an AhR antagonist revealed that the AhR pathway is required for the improved epithelial barrier functions of GarD. These results highlight a dual mechanism by GarD that confers protection against intestinal epithelial barrier dysfunction.

The onion-derived metabolite, S-1-propenylmercaptocysteine (CySSPe), protects against oxidative stress and exhibits anti-inflammatory effects by modulating cellular redox homeostasis. We sought to establish whether CySSPe activates nuclear factor erythroid 2-related factor 2 (Nrf2) and whether activation of Nrf2 by CySSPe involves modification of the Kelch-like ECH-associated protein-1 (Keap1) to manifest these effects. We found that CySSPe stabilized Nrf2 protein and facilitated nuclear translocation to induce expression of antioxidant enzymes, including NQO1, HO-1, and GCL. Moreover, CySSPe attenuated tert-butyl hydroperoxide-induced cytotoxicity and dose-dependently inhibited reactive oxygen species production. Silencing experiments using Nrf2-siRNA confirmed that CySSPe conferred protection against oxidative stress by activating Nrf2. CySSPe enhanced cellular pool of reduced glutathione (GSH) and improved GSH:GSSG ratio. Pretreatment of cells with l-buthionine-S,R-sulfoximine (BSO) confirmed that CySSPe increases de novo synthesis of GSH by upregulating expression of the GSH-synthesizing enzyme GCL. Treatment of cells with CySSPe elevated hydrogen sulfide (H2S) production. Inhibition of H2S-synthesizing enzymes, cystathionine-gamma-lyase (CSE) and cystathionine-beta-synthase (CBS), by pretreating cells with propargylglycine (PAG) and oxyaminoacetic acid (AOAA) revealed that H2S production was partially dependent on a CSE/CBS-catalyzed β-elimination reaction with CySSPe that likely produced 1-propenyl persulfide (RSSH). Depleting cells of their GSH pool by exposure to BSO and diethylmaleate attenuated H2S production, suggesting a GSH-dependent formation of H2S, likely via the reduction of RSSH by GSH. Finally, treatment of cells with CySSPe persulfidated Keap1, which may be the mechanism involved for the stabilization of Nrf2 by CySSPe. Taken together, our results showed that attenuation of oxidative stress by CySSPe is associated with its ability to produce H2S or RSSH, which persulfidates Keap1 and activates Nrf2 signaling. This study provides insights on the potential of CySSPe as an onion-derived dietary agent that modulates redox homeostasis and combats oxidative stress.

This data article contains experimental data on the preparation and semi-preparative isolation of S -Alk(en)ylmercaptocysteine (CySSRs, R = allyl, “A”, 1-propenyl, “Pe” or methyl, “Me”) generated through conjugation reactions between allyl and 1-propenyl enriched thiosulfinates (TS) and cysteine. The data presented are related to the research article “ S- Alk(en)ylmercaptocysteine suppresses LPS-induced pro-inflammatory responses in murine macrophages through inhibition of NF-κB pathway and modulation of thiol redox status” (Tocmo and Parkin, in press). In this data article, we included a detailed procedure for CySSR preparation, their purification through semi-preparative chromatography and their toxicity profiles in RAW 264.7 macrophages. Data included also highlight, the ability of CySSRs to modulate intracellular thiol redox status.

Buckwheat is an important alternative crop and a raw material for functional food formulation. Phase II detoxification proteins/enzymes provide cytoprotective roles against oxidative stress and inflammation originating from various stressors. We aimed to identify and characterize potential phase II enzyme inducers from methanolic extracts of buckwheat flour, using an activity-guided fractionation based on the induction of quinone reductase (QR) (EC 1.6.5.2) in Hepa 1c1c7 cells.; Results: We isolated the QR inducers N-trans-feruloyltyramine (I), syringic acid (II), quercetin (III) and myricetin (IV). The relative QR-inducing ability, as well as the concentration required to double QR specific activity (CD values, in parentheses), decreased in the order: quercetin (3.0 µmol L-1 ) > N-trans-feruloyltyramine (24 µmol L-1 ) > myricetin (58 µmol L-1 ) > syringic acid (5.4 mmol L-1 ). Quercetin and N-trans-feruloyltyramine exhibited the greatest extent of QR induction of an approximately four-fold maximum induction and these compounds also exhibited the greatest values for the ratio of IC50 (i.e. level to reduce viability by 50%): CD values of 11 and > 8.3, respectively, among the four QR inducers isolated. Isobologram analyses for binary combinations of compounds I-IV revealed primarily antagonistic interactions for QR induction.; Conclusion: These findings add to our understanding of the nutraceutical potential of buckwheat as a chemoprophylactic dietary component. © 2018 Society of Chemical Industry.; © 2018 Society of Chemical Industry.

The Allium vegetable-derived metabolite, S-alk(en)ylmercaptocysteine (CySSR), has been reported to modulate oxidative stress and inflammatory responses. However, the underlying mechanisms of action and structure-activity relationships are not completely understood. We investigated the mechanistic basis of the protective effects of CySSR on pro-inflammatory responses involving redox/oxidative stress induced by E. coli lipopolysaccharide (LPS) using RAW 264.7 cells. CySSR (R = allyl, “A” or 1-propenyl, “Pe”) pre-treatments conferred concentration-dependent reductions in cytokines (TNF-α, IL-1β and IL-6), NO production and iNOS (inducible nitric synthase) overexpression, and attenuated oxidant production in LPS-stimulated RAW 264.7 cells where viability remained > 90%. These protective effects were manifested through inhibited activation of the nuclear factor-kappa B (NF-κB) signaling pathway via suppression of the IκB kinases (IKK) phosphorylation possibly by transforming growth factor β-activated kinase 1 or a kinase further upstream the canonical NF-κB signaling pathway. The attenuation of LPS-induced inflammation by CySSRs was associated with enhanced levels of cellular cysteine (CySH) and glutathione (GSH) mediated by cellular import/reduction of CySSR and the induction of glutamate cysteine ligase (GCL), one of > 200 nuclear factor erythroid 2-related factor 2 (Nrf2) regulated proteins. The reduction of anti-inflammatory effect of CySSR following pretreatment of cells with L-buthionine-S,R-sulfoximine (BSO) implicates GSH having a major role in reducing inflammation, likely in the context of other Nrf2-regulated antioxidant enzymes that scavenge H2O2 and peroxides using GSH as co-substrate. The anti-inflammatory effect of CySSPe was significantly greater than CySSA for almost all indicators measured, and cell metabolites of CySSRs may have a role in attenuating NF-κB signaling.

Summary Methanolic extracts of bran from black glutinous rice cultivars were screened for potential health benefits using cellular bioassays for quinone reductase (QR) induction in murine hepatoma cells and inhibition of NO production in lipopolysaccharide (LPS)-activated RAW 264.7 macrophages. Fractionation of the crude extract by semi-preparative HPLC afforded respective fractions enriched in phenolic acids, anthocyanins and proanthocyanidins. The relative potency of QR induction was phenolic acids > proanthocyanidins > anthocyanins, and the activity of the crude extract was conserved among the aggregate of these fractions. In contrast, all three fractions were more potent than the crude extract in terms of anti-inflammatory effect. This suggests synergism effects among anti-inflammatory agents through partial purification. Inhibition of macrophage NO production by binary mixtures of gallic acid or caffeic acid with the anthocyanin-rich fraction indicated potential synergism. These results warrant further efforts to identify the active agents and substantiate synergistic interactions through isobologram analysis.

Buckwheat is an important alternative crop and a raw material for functional food formulation. Phase II detoxification proteins/enzymes provide cytoprotective roles against oxidative stress and inflammation originating from various stressors. We aimed to identify and characterize potential phase II enzyme inducers from methanolic extracts of buckwheat flour, using an activity-guided fractionation based on the induction of quinone reductase (QR) (EC 1.6.5.2) in Hepa 1c1c7 cells.We isolated the QR inducers N-trans-feruloyltyramine (I), syringic acid (II), quercetin (III) and myricetin (IV). The relative QR-inducing ability, as well as the concentration required to double QR specific activity (CD values, in parentheses), decreased in the order: quercetin (3.0 µmol LThese findings add to our understanding of the nutraceutical potential of buckwheat as a chemoprophylactic dietary component. © 2018 Society of Chemical Industry.

Introduction to Food Chemistry, O.R. Fennema, S. Damodaran, and K.L. Parkin Major Food Components Water and Ice, D.S. Reid and O.R. Fennema Carbohydrates, J.N. BeMiller and K.L. Huber Lipids, D.J. McClements and E.A. Decker Amino Acids, Peptides, and Proteins, S. Damodaran Enzymes, K.L. Parkin Minor Food Components Vitamins, J.F. Gregory III Minerals, D.D. Miller Colorants, S.J. Schwartz, J.H. von Elbe, and M.M. Giusti Flavors, R.C. Lindsay Food Additives, R.C. Lindsay Bioactive Substances: Nutraceuticals and Toxicants, C.T. Ho, M.M. Rafi, and G. Ghai Food Systems Dispersed Systems: Basic Considerations, P. Walstra and T. van Vliet Physical and Chemical Interactions of Components in Food Systems, Z.E. Sikorski, J. Pokorny, and S. Damodaran Characteristics of Milk, H.E. Swaisgood Postmortem Physiology of Edible Muscle Tissues, G. Strasburg, Y.L. Xiong, and W. Chiang Postharvest Physiology of Edible Plant Tissues, J.K. Brecht, M.A. Ritenour, N.F. Haard, and G.W. Chism Impact of Biotechnology on Food Supply and Quality, M. Newell-McGloughlin Appendices Appendix A: International System of Units (SI), The Modernized Metric System Appendix B: Conversion Factors (Non-SI Units to SI Units) Appendix C: Greek Alphabet Appendix D: Calculating Relative Polarities of Compounds Using Fragmental Constant Approach to Predict Log P Values. Index

Buckwheat is a raw material used in formulating many foods, and some evidence exists that it can contribute health benefits beyond simple nutritive value. The objective of this study was to isolate and identify potential cytoprotective agents from buckwheat crude extracts using a bioassay-guided strategy. Crude extracts were obtained by successively extracting with n-hexane and ethyl acetate, followed by solvent partitioning. The active fraction(s) were then screened by a quinone reductase (QR) induction bioassay in vitro, and subjected to sequential fractionation with flash column chromatography and preparative thin-layer chromatography. Consequently, three pure compounds were isolated, identified and evaluated for bioactivity. Ferulic acid ethyl ester (1) was the most potent isolate (reported for the first time in buckwheat), doubling QR specific activity (CD value) at 2.1 μM, whereas furaneol (2) was a moderate QR inducer with a CD value of 185 μM. Protocatechuic acid (3) was least ineffective at inducing QR with a CD value of 2.0 mM. Binary mixtures of the three isolated components acted borderline additively/antagonistically in the QR bioassay.

Ethanolic extracts of three different varieties of purple maize (Zea mays L.) were examined for scavenging activities toward nitric oxide (NO) and superoxide (·O2−) and the ability to induce quinone reductase (QR). The crude extracts showed a dose-dependent antioxidant activity in the following order: generic purple > Oaxaca 332 > Veracruz 42. The extract of the generic purple variety exhibited the highest ability to induce QR activity and antioxidant activity was split into several fractions that showed antioxidant activities and capacity for doubling QR specific activity. Fraction III doubled QR at a content of 105 μg/mL (CD value). Semi-preparative LC and UV spectral and MS analyses of fraction III indicated the presence of glucosidic forms of cyanidin, peonidin, pelargonidin and their respective acylated counterparts.

Microbial transformations of intrinsic substrates offer immense potential for generating new bioactive compounds in fermented food products. The aim of this work was to characterize the secondary metabolites in soy sauce, one of the oldest fermented condiments. Ethyl acetate extract (EAE) of soy sauce was separated using flash column chromatography, crystallized, and analyzed by nuclear magnetic resonance (NMR), single-crystal X-ray diffraction (SC-XRD), and mass spectroscopy. Dihydrocanadensolide (DHC), an antiulcer agent, was identified in a food for the first time. The natural stereostructure of DHC, which remained controversial for several decades, was determined as (3S,3aS,6R,6aR)-6-butyl-3-methyltetrahydrofuro[3,4-b]furan-2,4-dione using SC-XRD analysis. Kojic acid (KA) and vanillic acid (VA) were also identified from EAE as bioactive metabolic products of fungi and yeasts. Moreover, a new polymorphic form of KA was determined by SC-XRD.

S-Allylmercaptocysteine (CySSA) from garlic is known to exhibit anti-cancer effects. Apoptosis induction by CySSA was contrasted with S-1-propenylmercaptocysteine (CySSPe) (the major onion analog) in the presence of Na2SeO3 (Se) in breast cancer cells MCF-7. The dose of CySSA or CySSPe alone required to reduce viable cells by 50% was >400μM, and this was reduced to 62μM and 91μM for CySSA+Se and CySSPe+Se, respectively, at molar ratios of 39:1. Synergism of the mixtures was confirmed by isobologram analysis and the treatments evoked enhanced thiol efflux from MCF-7 cells. Apoptosis was confirmed by Annexin-V and propidium iodide staining. Cell cycle arrest occurred at the G2/M and sub-G1 interphases. Both CySSR+Se mixtures reduced the levels of Akt. CySSPe+Se elevated GSK-3 protein levels, whereas CySSA+Se did not. CySSR+Se mixtures enhanced phospho-c-Jun levels, with CySSA+Se more potent than CySSPe+Se. Corresponding increases in phospho-p53, Bax and Bad levels were observed, indicating apoptosis occurred via the mitochondrial pathway. Lack of caspases 6/7 activation implicated a caspase-independent pathway for apoptosis. Reduction of imported CySSR and export of thiols by MCF-7 cells facilitates the reduction of selenite to yield H2Se, a cytotoxic agent. This appears to be the first report of an anti-cancer effect of CySSPe.

6-Dehydroshogaol (6-DHSG) is a bioactive α,β-unsaturated carbonyl compound isolated from fresh ginger with anti-inflammatory and phase II enzyme inducing activities. Here we describe the glutathione (GSH)-dependent metabolism and the effect of this metabolic transformation on the biological activities of 6-DHSG. Compared with other ginger compounds, such as 6-gingerol and 6-shogaol, 6-DHSG showed the most potent anti-inflammatory effect in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. The biological activities of 6-DHSG were attenuated by sulfhydryl antioxidants such as glutathione (GSH) or N-acetyl cysteine (NAC), but not ascorbic acid (ASC). 6-DHSG was metabolised by GSH to form a GSH conjugate (GS-6-DHSG) in RAW 264.7 cells, via a potential mechanism involving the catalytic activity of glutathione-S-transferase (GST). GS-6-DHSG showed reduced biological activities compared with 6-DHSG in multiple biological assays. Together, these results indicate that GSH conjugation attenuates the biological activities of 6-DHSG and other α,β-unsaturated carbonyl compounds.

S-Alk(en)ylmercaptocysteine (CySSR, R = methyl, ethyl, propyl, 1-propenyl, and allyl), which are the putative metabolites of Allium thiosulfinates, were chemically synthesized. CySSR, but not the corresponding monosulfide species S-alk(en)yl cysteine (CySR), were able to induce quinone reductase (QR, a representative phase II enzyme) in Hepa 1c1c7 cells and inhibit nitric oxide (NO, an inflammatory biomarker) formation in lipopolysaccharide (LPS)-activated RAW 264.7 cells. These results indicate the importance of the disulfide bond for the biological activities of CySSR. Glutathione (GSH) and N-acetylcysteine (NAC), but not other types of cellular antioxidants, suppressed multiple biological activities of CySSR in vitro. The inhibitory effects of GSH and NAC on the biological activities of CySSR were correlated with a glutaredoxin (Grx)-dependent intracellular reduction of CySSR to generate cysteine and RSH, which were secreted into the extracellular medium.

Hexahydrocurcumin, 1-dehydro-[6]-gingerdione, 6-dehydroshogaol and 6-shogaol were evaluated for their antioxidant and anti-inflammatory activities in the present study. The relative antioxidant potencies of ginger compounds decreased in similar order of 1-dehydro-[6]-gingerdione, hexahydrocurcumin>6-shogaol>6-dehydroshogaol in both 1,1-diphenyl-2-picyrlhydrazyl (DPPH) radical-scavenging and trolox equivalent antioxidant capacity (TEAC) assays. All tested compounds could attenuate lipopolysaccharide (LPS)-elicited increase of prostaglandin E2 (PGE(2)) in murine macrophages (RAW 264.7) in a concentration-dependent manner but hexahydrocurcumin of 7μM and 6-shogaol of 7μM. The strongest inhibitory effect was observed for 6-dehydroshogaol and 6-shogaol at 14μM with the inhibition of 53.3% and 48.9%, respectively. Furthermore, both 6-dehydroshogaol and 1-dehydro-[6]-gingerdione significantly suppressed the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) proteins in a concentration-dependent fashion. These results contribute to our theoretical understanding of the potential beneficial effects of consuming ginger as a food and/or dietary supplement.

About 90–98% of native lycopene exists in the all- E form, but 79–88% of the lycopene found in the human body are Z -isomeric forms. Thermally-induced geometrical isomerisation of lycopene occurred within 24 h of refluxing in ethyl acetate and the proportion of Z -isomers increased from 5.8% to 49.9%. Accordingly, the concentration of lycopene required to double quinone reductase (QR) activity in Hepa 1c1c7 cells decreased from >100 to ∼22 μg/mL following thermo-isomerisation, while cell viability was retained at >87% at levels up to 50 μg/mL. The inhibition of nitric oxide (NO) production in activated RAW 264.7 macrophages was 50% at ∼100 μg/mL thermo-isomerised lycopene and increased to >80% when the concentration in the medium was increased to 500 μg/mL. No significant inhibition of NO evolution by macrophages occurred with native (∼94% all- E ) lycopene. Both QR induction and NO inhibition bioassays revealed that the structural changes evoked by thermo-isomerisation were accompanied by enhanced biological functionality.

Summary The effect of processing conventional and pigmented corn into masa, tortilla and tortilla chips on the ability to scavenge nitric oxide (NO) and peroxynitrite (ONOO−) was investigated. The level of retention of nitric oxide and peroxynitrite (mediated for inhibition of nitrite formation scavenging capacity for masa, tortilla and tortilla chips) ranged 56.2–78.2%, 67.4–45%, 40.3–62.0% (for nitric oxide) and 38.6–81.7%, 23.3–47.7%, 19.3–67.2% (for inhibition of nitrite formation), respectively. The antinitrosative activities were affected significantly (P > 0.05) by nixtamalisation, but not by processing masa into tortilla and tortilla chips (P > 0.05). The yellow variety and its corresponding products showed the greatest capacity to scavenge nitric oxide and inhibition of nitrite formation among conventional varieties and the purple variety ranked highest among the pigmented varieties.

To investigate the potential activity of ginger rhizome extracts to induce quinone reductase (QR), we performed bioactivity-guided fractionation using a murine hepatoma cell (Hepa 1c1c7) bioassay. Anti-inflammatory effects were then studied utilizing lipopolysaccharide (LPS)-stimulated mouse macrophage (RAW 264.7) cells. An ethyl acetate-partitioned fraction from ethanolic extract, rich in both QR inducing potency and anti-inflammatory activity, was subjected to repeated silica gel column and preparative thin layer chromatography to yield three compounds. The three isolated compounds were [6]-shogaol, 1-dehydro-[6]-gingerdione and hexahydrocurcumin. [6]-Dehydroshogaol, a minor component in ginger rhizome, was chemically synthesized and used for comparison in the subsequent bioassay based on its excellent QR inducing potency. Results showed that [6]-dehydroshogaol had the highest ability to induce QR activity (CD = 9.23 ± 0.22 μM), followed by 1-dehydro-[6]-gingerdione (CD = 13.24 ± 0.45 μM), and then hexahydrocurcumin (CD = 68.81 ± 3.90 μM). Increasing QR activity in induced cells was associated with elevated expression of NQO-1 protein as confirmed by Western blot. [6]-Dehydroshogaol, [6]-shogaol and 1-dehydro-[6]-gingerdione were also potent inhibitors of nitric oxide (NO) synthesis in activated macrophages. Their IC 50 values ranged from 5.80 ± 1.27 to 25.06 ± 4.86 μM. Hexahydrocurcumin exhibited the weakest inhibitory effect (IC 50 = 304.76 ± 54.80 μM). These findings contribute to our theoretical understanding of the potential beneficial effects of consuming ginger as food and/or dietary supplement.

A murine hepatoma (Hepa 1c1c7) cellular bioassay was used to guide the isolation of phase II enzyme inducers from fermented soy sauce, using quinone reductase (QR) as a biomarker. A crude ethyl acetate extract, accounting for 8.7% of nonsalt soluble solids of soy sauce, was found to double relative QR specific activity at 25 μg/mL (concentration required to double was defined as a "CD value"). Further silica gel column fractionation yielded 17 fractions, 16 of which exhibited CD values for QR induction of100 μg/mL. The four most potent fractions were subfractionated by column and preparative thin layer chromatography, leading to the isolation and identification of two phenolic compounds (catechol and daidzein) and two β-carbolines (flazin and perlolyrin), with respective CD values of 8, 35, 42, and 2 μM. Western blots confirmed that the increases in QR activity corresponded to dose-dependent increases in cellular levels of NAD[P]H:quinone oxidoreductase 1 protein by these four QR inducers. To the authors' knowledge, this is the first report on the ability of β-carboline-derived alkaloids to induce phase II enzymes.

Ferulic acid ethyl ester (FAEE) and ferulic acid (FA) were fed to rats to determine in vivo efficacy in elevating selected phase II enzyme activities and antioxidant capacity. Male Sprague–Dawley rats were fed the AIN-93M control diet, or the control diet supplemented with 1% FA, 1% FAEE, or 0.1% FAEE for 2 weeks. Quinone reductase (QR), glutathione-S-transferase (GST), thioredoxin reductase (TxR) activities, and oxidised and reduced glutathione were determined for brain, lung, liver, kidney, intestine and colon tissues. Both FA and FAEE (1%) supplementation increased QR and GST specific activities in kidney and colon tissues by 23–38% relative to the control diet. FAEE (1%) supplementation also induced QR specific activity, by 1.46- and 1.27-fold over the control diet, in intestinal and lung tissue of animals, whereas FA did not. No effect of diet was observed on liver cytochrome P450-1A1 activity. These results demonstrate that dietary FA and FAEE induce QR activity in the colon, small intestine, lung, and kidney, and improve glutathione antioxidant status in the colon and intestine. Therefore, dietary FA and FAEE may elevate defences to oxidative- and xenobiotic-induced stress in vivo.

Quinone reductase (QR) inducing activity, a biomarker for Phase II enzyme induction, was detected in a crude ginger rhizome ethanolic extract using a murine hepatoma cell (Hepa 1c1c7) bioassay. An ethyl acetate-partitioned fraction became enriched in QR inducing activity with a CD value (concentration required to double QR specific activity) of 7 μg/ml and was further separated by silica flash chromatography. The four major fractions recovered exhibited CD values ranging 10–20 μg/mL for QR inducing activity. 1 H NMR analysis revealed an abundance of gingerols in the first 3 fractions, and this led to a comparative assessment of QR inducing and anti-inflammatory activities among [6]-gingerol and structurally related vanilloids from ginger and other spices. [6]-Dehydroshogaol (DHSG), a minor component of fresh ginger, had QR inducing activity (CD = 8.4 μM) of comparable potency to the well known cancer preventive agent, curcumin (CD = 4.3 μM). In contrast, [6]-gingerol the major pungent component of ginger showed less potent QR inducing activity (CD = 55 μM) and capsaicin exhibited the weakest inducing activity (CD = 103 μM). [6]-Shogaol, [6]-DHSG and curcumin were also potent inhibitors of nitric oxide (NO) synthesis, inhibiting 50% NO evolution in activated macrophages at levels of 10–12 μM (IC 50 values). Comparatively, IC 50 values for [6]-gingerol and capsaicin were about an order of magnitude greater (less potent) at 90 μM and 169 μM, respectively. This appears to be the first report of biological activities of [6]-DHSG, and Phase II enzyme-inducing ability of shogoal and gingerol.

Whole cells of Natrinema gari BCC 24369, a novel halophilic archaeon, were immobilised on various matrices by different techniques. Among all matrices tested, Celite showed the highest immobilisation yield. The immobilised whole cells retained histamine-degrading activity as high as 94% of the original activity detected in free whole cells. The catalytic properties of the immobilised whole cells on the Celite support were similar to the corresponding free whole cells, including optimal NaCl concentration (4.0–5.0 M), optimal pH (6.5–7.5), and optimal temperature (40–55 °C). Histamine-degrading activity, either in the presence of NaCl at elevated concentrations or at elevated temperatures, became more stable due to immobilisation. Both free and immobilised whole cells were able to degrade histamine specifically. The immobilised whole cells could be reused for the degradation of histamine up to seven cycles without any significant loss in activity. The immobilised whole cells have the potential to be applied for the degradation of histamine in products high in salt.

The presence of high level of histamine is detrimental to the quality and safety of fish sauce. Therefore, this study aimed to study the ability of extremely halophilic archaea to reduce histamine under high salt condition and to examine the enzyme activity potentially involved. Of 156 extremely halophilic archaea isolated from various salt-fermented fishery products, HDS3-1 from an anchovy fish sauce sample fermented for 3 months, exhibited the highest histamine degradation activity when cultured in halophilic medium containing 5 mM histamine (free-base), followed by HDS1-1, HPC1-2, and HIS40-3, respectively. HDS3-1 was classified as Natrinema gari based on 16S rRNA gene sequence similarities and did not exhibit decarboxylase activity toward all tested amino acids. Based on in vitro cytotoxicity assay, the treatment with whole cell extract of HDS3-1 to all cell lines tested resulted in dose-dependent inhibitions of the cell growth with the IC50 values higher than 250 μg ml−1. Histamine-degrading activity of HDS3-1 was located in the intracellular fraction and required 1-methoxy-5-methylphenazinium methylsulfate (PMS) as an electron carrier. The optimal pH, salt concentration, and temperature for histamine degradation were pH 6.5–8, 3.5–5 M NaCl, and 40–55 °C, respectively. The activity was fully retained at pH 6.5–9, in the presence of NaCl above 2.5 M, and at temperature lower than 50 °C. The results suggested that histamine-degrading activity of HDS3-1 was most likely associated with salt-tolerant and thermo-neutrophilic histamine dehydrogenase.

The chemical syntheses of cysteine (CYS) and glutathione (GSH) mixed -disulfide conjugates (CySSR, GSSR, respectively) of mercapto residues representing most of the R groups of thiosulfinates (R = methyl, ethyl, propyl, and allyl) are described. Gram-scale conjugates were prepared as98% pure preparations, with 80% reaction yield for each of the two seminal synthesis steps, with structures confirmed by (1)H NMR and high-resolution MS analyses. These conjugates are derivatives of thiosulfinates that may be evolved in processed foods, in the digestive tract, and through in vivo metabolism. The prepared conjugates were found to be able to induce quinone reductase (QR, a representative phase II enzyme) in murine hepatoma cells (Hepa 1c1c7) and to inhibit nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated macrophage cells (RAW 264.7), indicating they have potential cancer preventive and anti-inflammatory activities. Among the prepared conjugates, the allyl conjugates of CYS and GSH, S-allylmercaptocysteine (CySSA) and S-allylmercaptoglutathione (GSSA), showed the most potent activity regarding QR induction and NO production inhibition. The conjugates with saturated R groups were also active and conferred biological activity as cystine and oxidized glutathione exhibited no effects in these cellular assays.

Free-radical scavenging, reducing activity and some phytochemical content (total phenolic, anthocyanin and ferulic acid) of eighteen Mexican maize phenotypes were determined. Total phenolic contents ranged from 215.8 to 3400.1 mg gallic acid/100 g of whole grain flour and total anthocyanins ranged from 1.54 to 850.9 mg cyanidin-glucoside equivalents/100 g of whole grain flour. Most of the phenolics in grain were in the bound form (ca. 85%), while anthocyanins were the major free phenolic compounds. Among the different samples, bound phenolic extracts of corn appeared to have greater anti-radical and reducing activities than free phenolic extracts from the same grain samples when tested at a normalized phenolic concentration. The phenotypes Veracruz 42 and AREQ516540TL exhibited the greatest activities and these purple-colored strains were most enriched in anthocyanins. Extracts from a red-colored phenotype Pinto were also among the most effective at exhibiting anti-radical activities. Differences in free-radical scavenging and reducing activities appeared to be dependent on the unique profile of anthocyanins and other phenolics in each phenotype.

Soy flour was successively extracted with ethyl acetate, ethanol, and water and tested for quinone reductase (QR) induction in the Hepa 1c1c7 cell line. The ethanol extract was found to be the most potent, with a CD value (concentration required to double QR specific activity) of 460 μg/ml. Phase separation of the ethanol extract and a phospholipid removal procedure yielded a fraction that doubled QR at 80 μg/ml. Reverse phase medium pressure chromatography of the purified ethanol extract yielded 13 fractions. Constituents of selected fractions were identified using mass spectrometry and NMR analysis. Fractions 2, 8, and 10 contained 47% of the recovered mass and were the least active of the isolates. These three fractions contained isoflavone glycosides. The latest eluting fractions 11–13 were found to be the most potent, and contained the majority of recovered inducing units (IUs), a measure of potency.

Aqueous extracts of green bean ( Phaseolus vulgaris L.) were examined for antioxidant functions in terms of total reducing power (TRP), peroxyl radical quenching (PRAC), total antioxidant activity (TAA), and the ability to induce quinone reductase (QR) as a biomarker for phase II detoxification enzymes in Murine hepatoma (Hepa) cells. In terms of Trolox equivalents (TE), crude aqueous extracts exhibited 71 μmol TE/g dry weight (gdw) for TRP, 29 μmol TE/gdw for PRAC, and 8.5 μmol TE/gdw for TAA. Aqueous extractable QR-inducing activity was fractionated by Sephadex LH-20 chromatography with several resulting fractions capable of doubling QR specific activity in the Hepa bioassay. Of 10 recovered fractions, the first and most polar fraction doubled QR at a level of 39 μg/ml (CD value), while the ninth fraction exhibited a similar CD value of 35 μg/ml. Semi-preparative LC and UV spectral analysis of fraction 9 indicated the presence of quercetin and kaempferol glycosides, and simple phenolics among the enriched QR-inducing isolates.

Breeders have found field screening for white rot (Sclerotium cepivorum Berk.) resistance in onion (Allium cepa L.) to be unreliable since consistently moderate to high disease levels that signifi cantly differentiate cultivars do not occur over fi eld sites and years. The objective was to determine if differences in onion white rot resistance levels were associated with differing S-alk(en)yl-L-cysteine sulfoxide (ACSO) levels. A collection of onion breeding lines and hybrids were evaluated in fi eld trials at six sites in 1999-2001. High performance liquid chromatography was used to analyze ACSOs in onion plant organs. Four main cysteine-sulfoxides exist in Allium L. species: methyl (MCSO), 2-propenyl (2-PeCSO), 1-propenyl (1-PeCSO), and propyl (PCSO). 1-PeCSO was predominant in onion leaves, bulbs, and roots. 2-PeCSO was found in trace amounts in onion leaves and roots. There was signifi cantly more 2-PeCSO and total ACSO (roots only) and 1-PeCSO (roots and bulbs) in accessions that were more susceptible to white rot in the fi eld trials. This is the fi rst report of signifi cant differences in ACSO contents among white rot susceptible and resis- tant onions. A covariance analysis was used to determine if the ACSO levels that signifi cantly distinguished among accessions could predict fi eld onion white rot reaction. 1-PeCSO from both roots and bulbs was the best predictor of fi eld disease incidence in fi eld sites that had low, moderate, and high disease levels. Although the ACSO concentrations were not assessed on an individual plant basis, breeders may be able to screen onions for resistance to S. cepivorum by comparing onion root or bulb 1-PeCSO levels based on the results from this research. White rot incidence in the fi eld should be higher in those plants whose roots and bulbs have the highest levels of 1-PeCSO. Sclerotium cepivorum, the cause of white rot, is a fungus that occurs in almost all Allium production areas and is a serious threat to the onion and garlic production worldwide. The specifi c interaction between the host and pathogen is partially understood. Sclerotium cepivorum sclerotia are held in dormancy by fun- gistasis (Allen and Young, 1968; Coley-Smith and King, 1969) until specifi c stimulation by volatile organosulfur compounds from Allium roots. This was determined using aqueous extracts made from Allium species and testing their effects on sclerotial germination (Coley-Smith and Holt, 1966). Volatile organosulfur compounds evolved from intact onion and leek (A. ampeloprasum L.) seedlings, condensed vapors evolved from garlic (A. sativum L.) juice, and distillates of garlic and onion juice also stimulated sclerotial germination (King and Coley-Smith, 1969). The Allium compounds that appear to be stimulatory are the organosulfur com- pounds that comprise fl avor in alliums. These compounds include thiosulfi nates, monosulfi des, disulfi des, trisulfi des, and thiophene derivatives (Coley-Smith and King, 1969; King and Coley-Smith, 1969), which are some of the products derived from the reaction between Allium fl avor precursors, S-alk(en)yl-L-cysteine sulfoxide (ACSO), and the enzyme alliinase (Block, 1992).

Fatty acid incorporation into discrete acylglycerol (AG) pools during esterification reactions with glycerol was analyzed for processes mediated by Rhizomucor miehei and Burkholderia cepacia lipases. Multicompetitive mixtures of saturated FA (C4–C18 even chain) and oleic acid (C18:1) were reacted with glycerol and the temporal incorporation of FA into mono-, di- and triacylglycerol pools (MAG, DAG, TAG) was quantified by relative selectivity constants and dimensionless fatty acid partitioning coefficients. Both analyses revealed a general fidelity of FA selectivity during FA incorporation into each AG pool for each lipase, and little evidence of preferential incorporation of C8–C18 FA into an individual AG pool was obtained. Exceptions were noted for C4–6 FA and C18:1 FA which became selectively diminished and enriched, respectively, in the DAG and TAG pools. The observation that FA selectivity may be generally conserved throughout the sequential steps of TAG assembly may simplify the design of processes designed to yield specifically structured TAG using these lipases.

An esterase gene, designated estB , was isolated from a genomic library of Lactobacillus casei LILA. Nucleotide sequencing of the estB gene revealed a 954-bp open reading frame encoding a putative peptide of 35.7 kDa. The deduced amino acid sequence of EstB contained the characteristic GXSXG active-site serine motif identified in most lipases and esterases. An EstB fusion protein containing a C-terminal 6-histidine tag was constructed and purified to electrophoretic homogeneity by affinity chromatography. The native molecular weight of EstB was 216.5±2.5 kDa, while the subunit molecular weight was 36.7±1.0 kDa. Optimum pH, temperature, and NaCl concentration for EstB were determined to be pH 7.0, 50 to 55°C, and 15% NaCl, respectively. EstB had significant activity under conditions simulating those of ripening cheese (pH 5.1, 10°C, and 4% NaCl). Kinetic constants (K M and V max ) were determined for EstB action on a variety of ethyl esters and ester compounds consisting of substituted phenyl alcohols and short n -chain fatty acids. For comparison purposes, EstA from Lb. helveticus CNRZ32 was purified to electrophoretic homogeneity and its substrate selectivity determined in a similar fashion. Different substrate selectivities were observed for EstB and EstA.

FA selectivity of a Celite-immobilized potato lipid acyl hydrolase (LAH) in esterification reactions with long-chain FA, including stearic acid (18∶0), oleic acid (18∶1), linoleic acid (18∶2), α-linolenic acid (18∶3), EPA (20∶5), and DHA (22∶6), and alcohol co-substrates (n-propanol, isopropanol, 1,3-propanediol, and glycerol) was studied in isooctane. Immobilized LAH was selective for FA of greater degrees of unsaturation (18∶3>18∶2>18∶1>18∶0) for all alcohol acceptors evaluated. Selectivity of LAH toward unsaturated C18 FA increased with an increase in water activity (a w ) from 0.19 to 0.90 for n-propanol, isopropanol, and 1,3-propanediol as alcohol co-substrates. In contrast, with glycerol as the alcohol cosubstrate, selectivity of LAH toward these unsaturated C18 FA increased with a decrease in a w from 0.90 to 0.19. In addition, immobilized LAH strongly discriminated against EPA and DHA for both 1,3-propanediol and glycerol as alcohol co-substrates.

Free-radical scavenging, reducing, and phase II enzyme-inducing activities of aqueous and 5% aqueous ethanol extracts of freeze-dried root tissue of four beet (Beta vulgaris L.) strains (red, white, orange, and high-pigment (red) phenotypes) were determined. Aqueous and ethanolic tissue extracts of the regular and high-pigment red phenotypes were most capable of inhibiting metmyoglobin/H2O2-mediated oxidation of 2-2‘-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) and 2,2‘-azobis-(2-amidinopropane) dihydrochloride (AAPH)-mediated bleaching of β-carotene. These same extracts were also most efficient at reducing ABTS radical cation and inducing quinone reductase in murine hepatoma (Hepa 1c1c7) cells in vitro. Keywords: Quinone reductase; phase II enzyme; antioxidant; chemopreventive; Hepa 1c1c7 cells; beetroot; betalains

Quinone reductase (QR)-inducing activity of aqueous and ethanolic extracts of common vegetables was determined using a murine hepatoma (Hepa 1c1c7) cell bioassay. QR induction observed for aqueous extracts reached 13-fold for corn, 8-fold for kale, and 5-fold for snap beans. QR induction of ethanol extracts reached about 3-fold for both garlic and snap beans, and about 2-fold for both red beetroots and kale. These aqueous extracts were effective inhibitors (kale ∼ red beetroots > corn ∼ green beans > garlic) of the generation of 2-2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) radical cation (ABTS•+) by metmyoglobin and H2O2, whereas the ethanolic extracts were not inhibitory. Aqueous extracts reduced pre-formed ABTS•+ more effectively (> 95% inhibition) than did ethanolic extracts (∼50 to 90% inhibition). All vegetable extracts inhibited oxyradical-mediated β-carotene bleaching, where kale and red beetroot extracts exhibited the strongest protective effect.

Pure thiosulfinates, R-S(O)S-R (2), where R = Me (2a), Pr (2b), or All (2c), at levels up to 4 mM were not capable of scavenging hydrogen peroxide or superoxide anion. Relative to standard antioxidants (ascorbic acid, n-propyl gallate, butylated hydroxytoluene, Trolox, and reduced glutathione), these thiosulfinates were 1-3 orders of magnitude less efficient at reducing 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical, 0.5-2 orders of magnitude less efficient at quenching singlet oxygen, and about equally effective at scavenging hydroxyl radical. Generally, AllS(O)SAll (2c) was the most effective and PrS(O)SPr (2b) was the least effective thiosulfinate in these assays, except that MeS(O)SMe (2a) exhibited no quenching effect toward singlet oxygen. These thiosulfinates were also incapable at levels up to 0.1 mM (where they were toxic) of in vitro induction of quinone reductase (QR) in murine hepatoma (hepa 1c1c7) cells. However, S-1-propenyl-L-cysteine sulfoxide (isoalliin, 1a) and cycloalliin (3) induced QR in this system at 2 mM and 1 mM, respectively, although doubling of QR required levels of 10-15 mM.

The effect of water activity (aw) and immobilization on fatty acid (FA) selectivity of Burkholderia (formerly Pseudomonas) cepacia, Rhizomucor miehei, Candida antarctica (type B), and Candida rugosa lipases in esterification reactions was determined. Studies were based on measuring ester formation in multicompetitive reaction mixtures containing either the homologous series of even carbon number n-chain saturated FA (C4-C18) or a series of n-chain (un)saturated FA (C18:X, where X = 0–3 double bonds) as cosubstrates with 1,3-propanediol in ter-butyl methyl ether at aw of 0.19, 0.69, and 0.90. Activity and FA selectively patterns were similar for free and Celite-adsorbed lipases in response to changes in aw, although specific effects were observed for selectivity of B. cepacia and C. rugosa lipases toward C16 and C4/C6 FA, respectively. Also, selectivity toward unsaturated C18:X FA as a group was modulated by changes in aw for three of the four lipase studied. Resin-fixed lipases from R. miehei and C. antarctica exhibited profound differences in activity and FA selectively in response to changes in aw, relative to free and Celite-bound forms. These findings suggest that FA selectivity for lipid modification is influenced by aw and immobilization, but that each lipase has a characteristic response to these factors in a manner that cannot be predicted. © 2001 John Wiley & Sons, Inc. Biotechnol Bioeng 75: 219–227, 2001.

Fatty acid (FA) selectivity of immobilized Candida antarctica B lipase was assessed as influenced by various cosubstrate systems for ester synthesis. Reaction mixtures contained a homologous series of even-chain n-acyl donor (C(4)(-)(16)) substrates (FA or their methyl esters, FAME) and a single alcohol cosubstrate (propanol, 2-propanol, or their acetate derivatives) in hexane. Multiple FA optima were often observed, with preferences for C(6) (or C(4)) followed by C(14) and sometimes C(10). The degree of selectivity among acyl donors was modest (up to 1.28-2.60, based on ratios of selectivity constants) and was dependent on the choice of cosubstrate system. Acyl group selectivity ranged up to 1.31-1.36 for [FA + alcohol], 1. 48-2.60 for [FAME + alcohol], 1.30-1.72 for [FA + alcohol acetate], and 1.28-1.88 [FAME + alcohol acetate] reaction systems. General shifts in selectivity were observed between short-chain (C(4)(-)(8)) and long-chain (C(10)(-)(16)) FA as groups with propanol cosubstrate, whereas shifts in reaction selectivity were observed toward specific FA(s) for 2-propanol cosubstrate. Selectivity among a series of alcohol cosubstrates ranged up to 13-fold in esterification reactions with C(6) FA.

Lipid acyl hydrolase (LAH; patatin) was purified from potato tubers by ammonium sulfate fractionation followed by anion-exchange and affinity chromatography. The major protein band of 40-43 kDa on SDS-PAGE appeared to be patatin, and it stained positive for lipase activity on native PAGE. Selectivity of a Celite-immobilized potato LAH in esterification reactions with n-acyl fatty acids (FA; C4, C6, C8, C10, C12, C14, C16, and C18) and alcohol acceptors (n-propanol, 2-propanol, 1,3-propanediol, and glycerol; 1,2-propanediol was not sufficiently reactive) was studied in isooctane. Immobilized LAH was highly selective for medium chain FAs (C8/C10) with a secondary optimum for chain lengths of C14/16. Water activity (a(w)) influenced activity and FA selectivity of the enzyme. Initial rates of ester synthesis were greatest at a(w) of 0.90 for all alcohol acceptors except for glycerol, where greatest initial rates were observed at a(w) of 0.19. Immobilized LAH preparations exhibited a bell-shape pH profile with optimum activity at pH 6-7 for ester synthesis, and no effect of pH on FA selectivity was observed.

Fatty acid [FA; butanoic (C4); octanoic (C8); tetradecanoic (C14); and cis-9,12-octadecadienoic (C18:2) acids] reaction selectivity and the corresponding acyl profiles in differentially accumulating acylglycerol (AG) products (mono-, di-, and triacylglycerols; MAG, DAG, TAG, respectively) were evaluated for Celite™-immobilized potato tuber lipid acyl hydrolase (LAH)-mediated esterification reactions in isooctane at 35°C and water activity of 0.19. The ordinal pattern of FA selectivities was C8>C14>C18:2>C4, and the AG products accumulating were α-MAG>DAG>β-MAG>TAG. A dimensionless expression for fatty acid partitioning coefficient (FAPC) was contrived to represent the partitioning patterns of specific FA into specific AG pools on the basis of an equivalent extent of FA reaction. These FAPC values indicated that preferential partitioning of FA was as follows: C4 was preferentially partitioned into TAG, DAG, and β-MAG; C8 was preferentially partitioned into DAG; C14 was preferentially partitioned into α,β-MAG; C18:2 was preferentially partitioned into α,β-MAG and TAG. These findings infer that the tendency for LAH-mediated esterifications to accumulate MAG is based, in part, on a constraint in reactivity of α-MAG of ≥10 acyl carbon groups to serve as acceptors for further esterification events. The general approach taken in this study may assist in identifying the discrete steps in assembling structured glycerides where different biocatalysts exhibit the greatest degree or control of reaction selectivity.

Esterification reaction selectivity in homogeneous organic media was evaluated for Candida antarctica B and Pseudomonas cepacia lipases toward members of an homologous series of even carbon number, saturated n -fatty acids (FAs) of 4–16 carbons (C4–C16), glycerol, and glycerol analogues ( n -propanol, isopropanol, 1,2-, and 1,3-propanediols). Dual FA optima were observed in almost all reaction mixtures, with C. antarctica and P. cepacia lipases generally favoring C10/C6 and C8/C16, respectively. Several explanations are offered to account for multiple FA optima. Reaction selectivity toward FAs was more influenced by steric than electronic features of FA substrates. Relative selectivity and discriminatory power among the series of FAs was modulated by the specific alcohol acceptor used. This may be founded on partial sharing of binding pockets by alcohol and FA co-substrates. Evidence was obtained that implicates the sn -2 alcohol functional group as being a determinant of FA selectivity and lipase discriminatory power.

The influence of immediate and delayed pH control, and added S-methyl-L-cysteine sulfoxide (MCSO) on the progress of pink discoloration in relation to the fate of thiosulfinates in extracts of yellow onion (Allium cepa) bulbs was evaluated over an incubation period of about 3 weeks at 21–24°C. Greatest discoloration occurred in extracts prepared with immediate pH control at 6.1, and least discoloration occurred at pH 3.2, over the pH range of 3.2–6.1. Initial thiosulfinate levels, in descending order of abundance, occurred at pH 6.1 > 5.0 > 4.2 > 3.2 for extracts subject to immediate pH control. However, over 21 days incubation, the lowest residual levels (ranging 20–60% of maximum levels) were observed for extracts adjusted to pH 3.2 and 6.1. Extracts subject to delayed (10 min after tissue disruption) pH control (initial extract pH of 5.2–5.4) displayed the greatest extent of discoloration, in descending order, at pH 4.0 > 5.0, 6.0 > 3.0. In these samples, initial thiosulfinate levels were similar and the greatest residual thiosulfinate levels over the 21-day incubation period were observed at pH 3.0 and 4.0 (about 50% of maximum levels), with the lowest residual levels observed for extracts adjusted to pH 6.0 (about 20% of maximum levels). Tissue extracts supplemented with 9- to 18-fold excess MCSO were subject to modest increases in both extent of discoloration and thiosulfinate levels.

A dipeptidase with prolinase activity from Lactobacillus helveticus CNRZ32, which was designated PepR, was purified to gel electrophoretic homogeneity and characterized. The NH2-terminal amino acid sequence of the purified protein had 96% identity to the deduced NH2-terminal amino acid sequence of the pepR gene, which was previously designated pepPN, from L. helveticus CNRZ32. The purified enzyme hydrolyzed Pro-Met, Thr-Leu, and Ser-Phe as well as dipeptides containing neutral, nonpolar amino acid residues at the amino terminus. Purified PepR was determined to have a molecular mass of 125 kDa with subunits of 33 kDa. The isoelectric point of the enzyme was determined to be 4.5. The optimal reaction conditions, as determined with Pro-Leu as substrate, were pH 6.0 to 6.5 and 45 to 50 degrees C. The purified PepR had a Km of 4.9 to 5.2 mM and a Vmax of 260 to 270 mumol of protein per min/mg at pH 6.5 and 37 degrees C. The activity of purified PepR was inhibited by Zn2+ but not by other cations or cysteine, serine, aspartic, or metal-containing protease inhibitors or reducing agents. Results obtained by site-directed mutagenesis indicated that PepR is a serine-dependent protease. Gene replacement was employed to construct a PepR-deficient derivative of CNRZ32. This mutant did not differ from the wild-type strain in its ability to acidify milk. However, the PepR-deficient construct was determined to have reduced dipeptidase activity compared to the wild-type strain with all dipeptide substrates examined.

Procedures were developed for the synthesis, purification and analysis of propanediol (PD) esters of n-fatty acids (FA). Mono- (MAPD) and di-acylated (DAPD) species were synthesized from PD and FA using an immobilized lipase (Candida antarctica B) in tert-butanol. MAPD and DAPD were isolated using silica gel column chromatography as approximately 95% pure preparations. Normal phase gradient LC provided for resolution of MAPD, DAPD and FA. UV220 nm detection provided a detection limit of about 1 microgram, and a linear response range of up to 2000 micrograms. Response factors were determined for MAPD, DAPD and FA components comprised of n-fatty acyl lengths of 4-16.

An endopeptidase gene (pepE) was isolated from a previously constructed genomic library of Lactobacillus helveticus CNRZ32. The pepE gene consisted of a 1,314-bp open reading frame encoding a putative peptide of 52.1 kDa. Significant identity was found between the deduced amino acid sequence of pepE and the sequences for aminopeptidase C from Lactobacillus delbrueckii subsp. lactis DSM7290, L. helveticus CNRZ32, Streptococcus thermophilus CNRZ302, and Lactococcus lactis subsp. cremoris AM2. A recombinant PepE fusion protein containing an N-terminal six-histidine tag was constructed and purified to electrophoretic homogeneity. Characterization of PepE revealed that it was a thiol-dependent protease having a monomeric mass of 50 kDa, with optimum temperature, NaCl concentration, and pH for activity at 32 to 37 degrees C, 0.5%, and 4.5, respectively. PepE had significant activity under conditions which simulate those of ripening cheese (10 degrees C, 4% NaCl, pH 5.1). PepE hydrolyzed internal peptide bonds in Met-enkephalin and bradykinin; however, hydrolysis of alpha-, beta-, and kappa-caseins was not detected.

Ten commercial sources of lipases were evaluated for comparative hydrolytic (in an aqueous emulsion) and ester synthesis (in microaqueous media) activities. There was little relationship between these activities for the lipases evaluated. An immobilized Rhizomucor miehei lipase had the greatest ester synthesis activity but had comparatively limited hydrolytic activity. For the five most effective ester synthesizing lipases, each had a characteristic optimum water content for maximum initial reaction rates. However, this optimum water content was greater than that associated with maximum reaction yield of ester formation, indicating the need for unique and differential moisture control during the progress of reaction to obtain maximum process efficiency.